Mutants capable of utilizing the pyrimidine biosynthetic intermediates carbamoylaspartate and dihydroorotate for growth were derived from pyrimidine auxotrophs of Salmonella enterica serovar Typhimurium LT2. The gain-of-function phenotypes both resulted from mutations in a single gene, yhiT, the third gene of a putative four-gene operon, yhiVUTS, for which there is no homologous region in Escherichia coli. Notably, when a mutant yhiT allele was transferred to a pyrimidine-requiring E. coli strain, the transformant was then capable of using carbamoylaspartate or dihydrorotate as a pyrimidine source. The operon arrangement of the yhiVUTS genes was supported by genetic analyses and studies employing RT-PCR, coupled to the determination of the transcriptional start site using 5′-random amplification of cDNA ends (RACE). Computer-generated predictions indicated that YhiT is an integral membrane protein with 12 putative transmembrane domains typical of bacterial transport proteins. Competition experiments showed that mutant YhiT interacts with the C4-dicarboxylates succinate and malate, as well as the amino acids aspartate and asparagine. The native function of wild-type YhiT remains undetermined, but the collective results are consistent with a role as a general transporter of C4-dicarboxylates and other compounds with a similar basic structure.